Antibody specifically binding to GLP-1R and fusion protein thereof with GLP-1

ABSTRACT

Disclosed in the present invention is an antibody specifically binding to GLP-1R and a fusion protein thereof with GLP-1. The fusion proteins can effectively bind to a human GLP-1R receptor and activate a receptor signaling pathway, thus are useful for treating diabetes, excessive weight, obesity and related disorders thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/911,715, which is a National Stage of International ApplicationNo. PCT/CN2014/083568, filed Aug. 1, 2014, which claims the benefit ofthe priority of Chinese Patent Application No. 201310350640.0, filedAug. 13, 2013; the disclosure of each of which is incorporated herein byreference in its entirety.

REFERENCE TO A SEQUENCE LISTING

The present specification is being filed with a Sequence Listing inComputer Readable Form (CRF), which is entitled14254-011-999_SEQLIST.txt of 70,497 bytes in size and was created Aug.10, 2018; the content of which is incorporated herein by reference inits entirety.

FIELD

The present invention relates to the technical field of antibodies,especially relating to an antibody specifically binding to GLP-1R andfusion proteins thereof with GLP-1.

BACKGROUND

Typical symptoms of type II diabetes include the following threeaspects: 1) the peripheral insulin resistance, mainly the responsivenessof bone and muscle to insulin is reduced, leading to affected glucoseoutput of these tissues (Kahn and Goldfine, J Diabetes Complication(1993) 7:92-105; Weyer et al., J Clin Invest. (1999) 104:787-794); 2)excessive hepatic glucose production, the regulation of liver cells tothe responsiveness of insulin is reduced (Kahn and Goldfine, J DiabetesComplication (1993) 7:92-105; Lam et al., Am J Physiol Endocrinol Metab.(2009) 11:375-378) and the excessive secretion of glucagon (Unger andOrci, Arch Intern Med. (1977) 137:482-491); and 3) disorders ofpancreatic islet beta cells, at an earlier stage of a disease, anincrease in beta cell proliferation and insulin secretion compensatesthe impact of insulin resistance on blood sugar (Bonner-Weir, TrendsEndocrinol Metab. (2000) 11:375-378), but with the increase of time andthe degree of insulin resistance, depletion of beta cells occurs,followed by decreased insulin secretion, thus leading to type IIdiabetes (DeFronzo, Diabetes. (1988) 37:667-687; Kahn et al., J Nutr.(2001) 131:354S-360S).

Glucagon like peptide-1 (GLP-1) is a peptide containing 30 amino acids.It is secreted from L intestinal cells in response to the intake ofglucose (Orskov et al., Diabetes (1994) 43:535-539; Drucker et al.,Proc. Natl. Acad. Sci. USA (1987) 84:3431-3438). After the secretionupon stimulation, GLP-1 binds to pancreatic GLP-1R (glucagon likepeptide-1 receptor) to activate the downstream adenylate cyclasesignaling pathway to promote the synthesis and secretion of insulin.GLP-1 secretion also reduces gastric emptying, thereby reducing theamount of glucose into the circulatory system after food digestion(Wettergren et al., Dig. Dis. Sci. (1993) 38:665-673). In mice and inpatients with type I and type II diabetes, GLP-1 increases insulinsecretion and reduces blood sugar concentration (Nauck et al., Diabetes.(1997) 105:187-195; Todd et al., Eur J Clin Invest. (1997) 27:533-536).Studies have shown that GLP-1 can also inhibit apoptosis of pancreaticbeta cells and promote their proliferation (Perfetti et al.,Endocrinology (2000) 141:4600-4605; Hui et al., Endocrinology (2003)144:1444-1455). The feasibility and efficacy of GLP-1 for the treatmentof diabetes patients have been proved clinically (Samson and Garber,Curr Opin Endocrinol Diabetes Obes. (2013) 20:87-97). There are alsopatents (U.S. Pat. No. 5,899,883 and U.S. Pat. No. 6,989,148) disclosingmethods for the treatment of diabetes by using GLP-1 and itsderivatives. However, GLP-1 has a short half-life in vivo and does nothave good therapeutic effects.

SUMMARY

One objective of the present invention is to provide an antibodyspecifically binding to GLP-1R.

The second objective of the present invention is to provide a fusionprotein of an antibody specifically binding to GLP-1R with GLP-1, whichcan extend the half-life of GLP-1 in vivo to retain the biologicalactivity of GLP-1. At the same time, the fusion protein formed by GLP-1and the antibody specifically binding to GLP-1R has the moleculartargeting properties provided by the antibody. Furthermore, theimmunogenicity of the antibody is also lower than that of other fusionpartners.

To solve the technical problems mentioned above, the present inventionprovides the following technical solutions.

An antibody specifically binding to GLP-1R comprises an amino acidsequence selected from:

(a) a light chain CDR3 sequence selected from:

light chain CDR3 sequences differing by no more than three amino acidadditions, substitutions and/or deletions in total from one of L1-L13light chain CDR3 sequences: SEQ ID NO: 46 to SEQ ID NO: 53; preferably,light chain CDR3 sequences differing by no more than two amino acidadditions, substitutions and/or deletions in total from one of L1-L13light chain CDR3 sequences: SEQ ID NO: 46 to SEQ ID NO: 53; and morepreferably, light chain CDR3 sequences differing by one amino acidaddition, substitution and/or deletion from one of L1-L13 light chainCDR3 sequences: SEQ ID NO: 46 to SEQ ID NO: 53;

(b) a heavy chain CDR3 sequence selected from:

heavy chain CDR3 sequences differing by no more than four amino acidadditions, substitutions and/or deletions in total from one of H1-H13heavy chain CDR3 sequences: SEQ ID NO: 20 to SEQ ID NO: 27; preferably,heavy chain CDR3 sequences differing by no more than three amino acidadditions, substitutions and/or deletions in total from one of H1-H13heavy chain CDR3 sequences: SEQ ID NO: 20 to SEQ ID NO: 27; morepreferably, heavy chain CDR3 sequences differing by no more than twoamino acid additions, substitutions and/or deletions in total from oneof H1-H13 heavy chain CDR3 sequences: SEQ ID NO: 20 to SEQ ID NO: 27;and further preferably, heavy chain CDR3 sequences differing by oneamino acid addition, substitution and/or deletion in total from one ofH1-H13 heavy chain CDR3 sequences SEQ ID NO: 20 to SEQ ID NO: 27; and

(c) a light chain CDR3 sequence from (a) and a heavy chain CDR3 sequencefrom (b).

Preferably, the antibody further comprises one or more amino acidsequences selected from:

(a) a light chain CDR1 sequence selected from:

light chain CDR1 sequences differing by no more than three amino acidadditions, substitutions and/or deletions from one of L1-L13 light chainCDR1 sequences: SEQ ID NO: 28 to SEQ ID NO: 37; preferably, light chainCDR1 sequences differing by no more than two amino acid additions,substitutions and/or deletions in total from one of L1-L13 light chainCDR1 sequences: SEQ ID NO: 28 to SEQ ID NO: 37; and more preferably,light chain CDR1 sequences differing by one amino acid addition,substitution and/or deletion from one of L1-L13 light chain CDR1sequences: SEQ ID NO: 28 to SEQ ID NO: 37;

(b) a light chain CDR2 sequence selected from:

light chain CDR2 sequences differing by no more than two amino acidadditions, substitutions and/or deletions from one of L1-L13 light chainCDR2 sequences: SEQ ID NO: 38 to SEQ ID NO: 45; and preferably, lightchain CDR2 sequences differing by one amino acid addition, substitutionand/or deletion from one of L1-L13 light chain CDR2 sequences SEQ ID NO:38 to SEQ ID NO: 45;

(c) a heavy chain CDR1 sequence selected from:

heavy chain CDR1 sequences differing by no more than two amino acidadditions, substitutions and/or deletions from one of H1-H13 heavy chainCDR1 sequences: SEQ ID NO: 6 to SEQ ID NO: 12; and preferably, heavychain CDR1 sequences differing by one amino acid addition, substitutionand/or deletion from one of H1-H13 heavy chain CDR1 sequences: SEQ IDNO: 6 to SEQ ID NO: 12; and

(d) a heavy chain CDR2 selected from:

heavy chain CDR2 sequences differing by no more than three amino acidadditions, substitutions and/or deletions from one of H1-H13 heavy chainsequences: SEQ ID NO: 13 to SEQ ID NO: 19; preferably, heavy chain CDR2sequences differing by no more than two amino acid additions,substitutions and/or deletions in total from one of H1-H13 heavy chainCDR2 sequences: SEQ ID NO: 13 to SEQ ID NO: 19; and more preferably,heavy chain CDR2 sequences differing by one amino acid addition,substitution and/or deletion from one of H1-H13 heavy chain CDR2sequences: SEQ ID NO: 13 to SEQ ID NO: 19.

An antibody specifically binding to GLP-1R comprises an amino acidsequence selected from:

(a) one or more light chain variable regions selected from:

i. light chain CDR1 sequences: SEQ ID NO: 28 to SEQ ID NO: 37;

ii. light chain CDR2 sequences: SEQ ID NO: 38 to SEQ ID NO: 45; and

iii. light chain CDR3 sequences: SEQ ID NO: 46 to SEQ ID NO: 53;

(b) one or more heavy chain variable regions selected from:

i. heavy chain CDR1 sequences: SEQ ID NO: 6 to SEQ ID NO: 12;

ii. heavy chain CDR2 sequences: SEQ ID NO: 13 to SEQ ID NO: 19; and

iii. heavy chain CDR3 sequences: SEQ ID NO: 20 to SEQ ID NO: 27; and

(c) a light chain variable domain sequence from (a) and a heavy chainvariable domain sequence from (b).

An antibody specifically binding to GLP-1R comprises an amino acidsequence selected from:

(a) a light chain variable region selected from:

i. amino acid sequences that are at least 80% identical to any of L1-L13light chain variable region sequences: SEQ ID NO: 81, SEQ ID NO: 83, SEQID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93,SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO:103, SEQ ID NO: 105; and

ii. amino acid sequences encoded by polynucleotide sequences that are atleast 80% identical to any of the polynucleotide sequences encoding forL1-L13 light chain variable region sequences: SEQ ID NO: 80, SEQ ID NO:82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ IDNO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQID NO: 102, SEQ ID NO: 104;

(b) a heavy chain variable domain sequence selected from:

i. amino acid sequences that are at least 80% identical to any of H1-H13heavy chain variable region sequences: SEQ ID NO: 55, SEQ ID NO: 57, SEQID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO: 79; and

ii. an amino acid sequences encoded by polynucleotide sequences that areat least 80% identical to any of the polynucleotide sequences encodingfor H1-H13 heavy chain variable region sequences: SEQ ID NO: 54, SEQ IDNO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74,SEQ ID NO: 76, SEQ ID NO: 78; and

(c) a light chain variable region sequence from (a) and a heavy chainvariable region sequence from (b).

Preferably, the antibody further comprises an amino acid sequenceselected from:

(a) L1-L13 light chain variable region sequences: SEQ ID NO: 81, SEQ IDNO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101,SEQ ID NO: 103, SEQ ID NO: 105;

(b) H1-H13 heavy chain variable region sequences: SEQ ID NO: 55, SEQ IDNO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75,SEQ ID NO: 77, SEQ ID NO: 79; and

(c) a light chain variable region sequence from (a) and a heavy chainvariable region sequence from (b).

Preferably, the combination (c) of a light chain variable regionsequence (a) and a heavy chain variable region sequence (b) is selectedfrom L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10,L11H11, L12H12, L13H13.

Preferably, the antibody also comprises an amino acid sequence selectedfrom:

(a) light chain constant region amino acid sequence: SEQ ID NO 106;

(b) light chain constant region amino acid sequence: SEQ ID NO 107;

(c) heavy chain constant region amino acid sequence: SEQ ID NO 108;

(d) heavy chain constant region amino acid sequence: SEQ ID NO 109;

(e) light chain constant region amino acid sequence: SEQ ID NO 106 andheavy chain constant region amino acid sequence: SEQ ID NO 108;

(f) light chain constant region amino acid sequence: SEQ ID NO 107 andheavy chain constant region amino acid sequence of SEQ ID NO 108;

(g) light chain constant region amino acid sequence: SEQ ID NO 106 andheavy chain constant region amino acid sequence: SEQ ID NO 109; and

(h) light chain constant region amino acid sequence: SEQ ID NO 107 andheavy chain constant region amino acid sequence: SEQ ID NO 109.

Preferably, the antibody is selected from murine antibodies, humanantibodies, humanized antibodies, chimeric antibodies, monoclonalantibodies, polyclonal antibodies, recombinant antibodies,antigen-binding antibody fragments, single-chain antibodies,double-chain antibodies, triple-chain antibodies, tetra-chainantibodies, Fab fragments, F(fa′)x fragments, domain antibodies, IgDantibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2antibodies, IgG3 antibodies, and IgG4 antibodies.

A GLP-1 fusion protein comprising GLP-1 and an antibody of the presentinvention, wherein GLP-1 comprises an amino acid sequence selected fromSEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQID NO:127.

Preferably, GLP-1 is fused with the light chain and/or heavy chain ofthe antibody of the present invention via N′—R1-L-R2-C′, N′—R2-L-R1-C′or N′—R2-R1_(r)-C′;

wherein L is a peptide linker sequence, comprising a full-length,partial or repeated amino acid sequence selected from LK1-LK3 amino acidsequences: SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112;

R1 is an amino acid sequence of GLP-1;

R1_(r) is a reverse amino acid sequence of GLP-1;

R2 is an amino acid sequence of the light chain or heavy chain of theantibody of the present invention;

C′ represents the hydroxyl terminal of the GLP-1 fusion proteinpolypeptide chain;

N′ represents the amino terminal of the GLP-1 fusion protein polypeptidechain.

A polynucleotide encodes a GLP-1 fusion protein of the presentinvention.

A vector comprises a polynucleotide of the present invention.

A host cell comprises a vector of the present invention.

A pharmaceutical composition comprises a GLP-1 fusion protein of thepresent invention and a pharmaceutically acceptable carrier.

Use of a pharmaceutical composition comprising or based on an antibodyor GLP-1 fusion protein of the present invention in the preparation of amedicament for preventing or treating non-insulin-dependent diabetes isdisclosed.

Given that the key role of GLP-1R plays in the use of glucagon likepeptide-1 for the regulation and control of blood glucose levels in typeII diabetes patients, and that its significant therapeuticcharacteristic is the ability of stimulating insulin secretion withoutthe associated risk of hypoglycaemia. GLP-1 is fused with an antibodyspecifically binding to GLP-1R in the present invention, therebyprolonging the half-life of GLP-1 in vivo to retain the biologicalactivity of GLP-1. At the same time, the fusion protein formed by GLP-1and the antibody specifically binding to GLP-1R has the moleculartargeting properties provided by the antibody. Furthermore, theimmunogenicity of the antibody is also lower than that of other fusionpartners.

The beneficial effects of the present invention are as follows: GLP-1 iscapable of fusing with an antibody specifically binding to GLP-1R, thusprolonging the half-life of GLP-1 in vivo to retain the biologicalactivity of GLP-1. At the same time, the fusion protein formed by GLP-1and the antibody specifically binding to GLP-1R has the moleculartargeting properties provided by the antibody. Furthermore, theimmunogenicity of the antibody is also lower than that of other fusionpartners.

DETAILED DESCRIPTION

The present invention is directed to the disadvantage that GLP-1 isquickly removed by dipeptidyl peptidase (DPP-IV) in vivo and hasinsufficient efficacy, and applies antibodies of GLP-1R to fuse withGLP-1 so as to enhance the half-life and biological activity of GLP-1.Antibodies used for the fusion do not hinder the binding of GLP-1 withreceptors, can specifically facilitate the biological activity ofGLP-1R, and due to their high affinity to receptors and stability, arecapable of enhancing the long lasting local concentrations of GLP-1around the receptors and thereby significantly increasing its effectivetime and potency for binding to the receptor. At the same time, thefusion with the antibody increases the steric hindrance for DPP-IV torecognize or capture GLP-1, thus reducing the elimination rate of GLP-1in vivo and increasing the effective time of GLP-1. According toliterature reports (Lin and Wang, J of Molecular Modeling (2009)15:53-65), the release of the articulation state between the N-terminalextracellular region of GLP-1R and the transmembrane region thereof isan essential step for GLP-1 to enter the binding site to GLP-1R andbecome biologically active. As described in the present invention, thebinding of the antibody to GLP-1R is largely involved in the N-terminalextracellular region of the receptor, and the binding thereof to thereceptor helps the release of said articulation state and can facilitatethe access of GLP-1. Accordingly, the fusion of GLP-1 with the antibodytargeting GLP-1R increases the half-life and affinity potency of GLP-1to result a stronger biological activity, and thus is an importantinnovation superior to the GLP-1 therapy. More importantly, some of theantibodies themselves used in the present invention have the biologicalcharacteristics of enhancing GLP-1 activation of GLP-1R in the presenceof GLP-1. Because of some of the reasons above, the GLP-1 fusion proteinin the present invention may be a more effective activator of GLP-1Rthan GLP-1.

It is a common concern that the repetitive administration of a fusionprotein for a long time may elicit antigenicity. This is especially aconcern in the case of GLP-1 fusion protein therapy, because once apatient is diagnosed with diabetes, the patient is to receive alife-long treatment for the disease. In addition, if the Fc parts of theimmune globulin retain undesirable effector function, the Fc fusionprotein therapy can be a concern. Via computer-assisted 3D structureprediction of an immune globulin, and antibody sequence optimization andhumanization, the identified specific GLP-1 fusion proteins no longerhave effector function and thus have reduced risk of inducing immuneresponse after repeated and long-term administration. As discussed inthe present invention, the amino acid of GLP-1 moiety is preferablyfused with light and heavy chains of the antibodies through glycine andserine rich peptide linker. Because of having smaller side chains,glycine and serine enable the peptide linker sequence considerablyflexible, reducing the rigidity between GLP-1 and the correspondingpositions of the antibody, thus GLP-1 can interact with GLP-1R freely.At the same time, the presence of the peptide linker separates GLP-1from the antibody, thus avoiding the interaction of the two domains. Theglycine and serine appear alternately to avoid excessive repetition, inorder not to introduce undesirable immunogenicity to the fusion protein,however, the peptide linker inevitably increases the immunogenicity ofthe fusion protein in vivo, and it is of great importance to select thelength of the peptide linker so as to balance structure flexibility andimmunogenicity. Accordingly, the present invention provides threedifferent lengths of peptide linkers for fusion. At the same time, thepresent invention provides different ways of linking GLP-1 with theantibody by using peptide linkers for fusion, and the patterns of theformed GLP-1 fusion proteins would include:

1) a fusion protein with GLP-1 and a light chain linked in the form ofN′—R1-L-R2-C′;

2) a fusion protein with GLP-1 and a light chain linked in the form ofN′—R2-L-R1-C′;

3) a fusion protein with GLP-1 and a light chain linked in the form ofN′—R2-L-R1r-C′;

4) a fusion protein with GLP-1 and a heavy chain linked in the form ofN′—R1-L-R2-C′;

5) a fusion protein with 1) and 4) at the same time;

6) a fusion protein with 2) and 4) at the same time;

7) a fusion protein with 3) and 4) at the same time.

Within the scope of the present invention, the DNA encoding the GLP-1 islinked to full length/variable region/fragment light chain or fulllength/variable region/full length heavy chain DNA of said antibody, viathe DNA encoding the peptide linker sequence, forming a fused lightchain or fused heavy chain DNA, furthermore, at the 5′ end of the lightchain DNA, the DNA encoding the signal peptide is also introduced toform a gene based on which the mutant/wild type GLP-1 can be linked toantibody sequences. In the present invention, the GLP-1 sequencesobtained by the method of gene synthesis are linked to the peptidelinker as well as antibody light or heavy chain DNA through the methodof PCR. The light or heavy chain variable region sequences of theantibodies to GLP-1R are obtained from specific hybridoma cells throughthe method of PCR followed by being linked to the constant region DNA ofspecific antibody subtype. The constant region DNA of the wild typeantibody subtype can be obtained from a specific clone library and usedas the basis of sequence optimization. After cloned into an expressionvector, genes used for expressing the fusion protein described hereinare used for producing and expressing the fusion proteins. After thelight chain and heavy chain expression vectors are paired duringexpression, the DNA carrying the genes are co-transfected or transformedinto a host cell. The promoter is induced by optimal adaption. Thetransformants or genes for amplifying desired sequences are cultured ina proper medium at an appropriate pH and temperature. DNA is usuallyintroduced by commonly used methods, such as CaPO₄, electroporation, andPEI etc.

The suitable host cells, suitable for the expression of the nucleic acidwithin the vector described herein, include higher eukaryotic cells, andthe examples of mammalian host cell line for expression include Chinesehamster ovary cell line (CHO) and human embryonic kidney cell (HEK293,or HEK293 cell line cultured in a suspension), and the signal peptide atthe N-terminal of the light chain guides the secretion of recombinantfusion proteins from the mammalian host cell line. The vector forexpression or cloning carries the selection marker that enables itscontinuously replication in host cells and is used to screen cellscapable of integrating the fusion protein encoding nucleic acid, andpromoter that effectively links with the fusion protein encodingsequence and guides mRNA synthesis. One example is to use a vectorcarrying antibiotics resistance and Hepatitis B virus and simian viruspromoter (SV40) to select CHO host cells stably expressing the fusionproteins.

After host cell lines have expressed fusion proteins, the presentinvention adopts an affinity chromatographic method to purify the partsecreted thereby in the cell culture supernatant. In an example of theinvention, the fusion protein fused with full-length antibody iscaptured by a protein G affinity chromatography column and then elutedfrom the chromatography column by low pH followed by collection. Mildelution conditions help to prevent denaturing of the protein.

The fusion protein of the present invention can be formulated with oneor more excipients. The fusion protein of the present invention can becombined with a pharmaceutically acceptable buffer having adjusted pHthat provides acceptable stability and is suitable for administration(such as parenteral administration). Optionally, one or morepharmaceutically acceptable antimicrobial reagents may be added.Preferred pharmaceutically acceptable antimicrobial reagents arem-cresol and phenol. One or more pharmaceutically acceptable saltsolution can be added to adjust the ionic strength or tension. One ormore excipients may be added to further adjust the isotonicity of theformulation. Glycerin is an example of excipients for adjusting theisotonicity. “Pharmaceutically acceptable” means being suitable foradministration to human or other animals, and therefore free of toxicingredients or undesirable pollutants and not interfere with theactivity of the active compounds therein.

The fusion protein of the invention can be prepared in a solutionpreparation or in a lyophilized powder that can be reconstituted withappropriate diluent. Lyophilized dosage form is one of the formulationtypes in which the fusion protein is stable, with or without thebuffering capacity to maintain the pH over its intended in-useshelf-life of the reconstituted product. The solution comprising fusionproteins discussed herein is preferably isotonic before lyophilizationto enable the formation of an isotonic solution after reconstitution.

A pharmaceutically acceptable salt form of the fusion proteins of thepresent invention is within the scope of the present invention. Commonlyemployed acids to form acid addition salts are inorganic acids, such ashydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, andphosphoric acid; and organic acids such as p-toluenesulfonic acid,methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, and acetic acid.Preferable acid addition salts are those formed with inorganic acids,such as hydrochloric acid and hydrobromic acid.

Base addition salts include those derived from inorganic bases, such asammonium, base or alkali earth metal hydroxide, carbonate, andbicarbonate. Such bases useful in preparing the salt solution of thepresent invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, and the like.

The fusion proteins of the present invention have biological activity.Biological activity refers to the ability of the fusion proteins to bindand activate the GLP-1R in vivo and stimulate stress response. Responsesinclude but not limited to, increased secretion of insulin, suppressionof secretion of glucagon, inhibition of appetite, weight loss, inductionof satiety, inhibition of apoptosis, and induction of pancreatic betacell proliferation and pancreatic beta cell differentiation. A number ofrepresentative GLP-1 fusion proteins are tested for in vitro and in vivoactivities. First, step 4 (FIG. 1) provides data on a fluorescencedetection assay of the fusion protein to interact with the GLP-1R. Then,step 12 provides in vitro activity test of the fusion proteininteracting with and activating human GLP-1R. In this set ofexperiments, CHO cells over-expressing human GLP-1R were used.Activation of the GLP-1R in these cells causes adenylyl cyclaseactivation which in turn induces expression of a reporter gene driven bya cAMP response element (CRE). Step 12 (FIG. 2) provides the data wherethe reporter gene is luciferase. In vitro experimental data indicatethat the fusion proteins are capable of binding and activating GLP-1Rand appear to be more effective than the native GLP-1 in vitro. Step 13(FIG. 3) provides the data of blood glucose concentration change of themice 16 hours (hr) after being intraperitoneally administrated with oneof the fusion proteins of the present invention. The in vivo datagenerated on mice of step 13 demonstrate the activity of the fusionprotein and its longer half-life than the native GLP-1.

Administration of the fusion protein may be via any route known to beeffective by the physician of ordinary skill. Peripheral parenteraladministration is one of such methods. Parenteral administration iscommonly understood in medical literature as the injection of a dosageform into the body with a sterile syringe or other mechanical devicesuch as an infusion pump. Peripheral parenteral routes includeintravenous, intramuscular, subcutaneous, and intraperitoneal routes ofadministration.

The fusion proteins of the present invention can also be administratedby oral, rectal, nasal, or lower respiratory routes, which arenon-parenteral routes. Of these non-parenteral routes, the lowerrespiratory route and the oral route are preferred.

The fusion proteins of the present invention can be used to treat a widevariety of diseases and conditions. The fusion proteins of the presentinvention primarily exert their biological effects by acting at GLP-1R.Subjects with diseases and/or conditions that respond favorably toGLP-1R stimulation or to the administration of GLP-1 compounds cantherefore be treated with the GLP-1 fusion proteins of the presentinvention. These subjects are referred to as subjects “in need oftreatment with GLP-1 compounds” or “in need of GLP-1R stimulation”.Included are subjects with non-insulin dependent diabetes, insulindependent diabetes, stroke (see WO 00/16797), myocardial infarction (seeWO 98/08531), obesity (see WO 98/19698), catabolic changes after surgery(see U.S. Pat. No. 6,006,753), functional dyspepsia and irritable bowelsyndrome (see WO 99/64060). Also included are subjects requiringprophylactic treatment with a GLP-1 compound, e.g., subjects at risk ofdeveloping non-insulin dependent diabetes (see WO 00/07617). Subjectswith impaired glucose tolerance or impaired fasting glucose, subjectswhose body weight is about 25% above normal body weight for thesubject's height and body fluid, subjects with a partial pancreatectomy,subjects having one or both parents with non-insulin dependent diabetes,subjects who have had gestational diabetes and subjects who have hadacute or chronic pancreatitis are at risk of developing non-insulindependent diabetes. An effective amount of the fusion proteins describedherein is the dosage which results in a desired therapeutic and/orprophylactic effect without causing unacceptable side-effects whenadministrated to a subject in need of GLP-1 receptor stimulation. A“desired therapeutic effect” includes one or more of the followings: anamelioration of the symptom(s) associated with the disease or condition;a delay in the onset of symptoms associated with the disease orcondition; increased longevity compared with the absence of thetreatment; and better quality of life compared with that in the absenceof the treatment. An “effective amount” of the GLP-1 fusion proteins forthe treatment of diabetes is the amount that would result in bettercontrol of blood glucose concentration compared with that in the absenceof the treatment, thereby resulting in a delay in the onset of diabeticcomplications such as retinopathy, neuropathy or kidney diseases. An“effective amount” of the GLP-1 fusion protein for the prevention ofdiabetes is the amount that would delay, compared with that in theabsence of treatment, the onset of elevated blood glucose levels thatrequires treatment with anti-hyperglycaemic drugs such as sulfonyl urea,thiazolidinedione, insulin and/or bisguanidine. The dosage of fusionproteins effective to normalize a patient's blood glucose will depend ona number of factors, among which are included, without limitation, thesubject's sex, weight and age, the severity of inability to regulateblood glucose, the route of administration and bioavailability, thepharmacokinetic profile of the fusion protein, the potency, and theformulation. Doses may be in the range of 0.01 to 1 mg/kg body weight,preferably in the range of 0.05 to 0.5 mg/kg body weight. It ispreferable that the fusion proteins of the present invention areadministered either once a week or twice a week. Depending on thedisease to be treated, it may be necessary to administrate the fusionprotein more frequently such as three or more times per week.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow cytometry (FACS) showing the specific binding of arecombinant expressed GLP-1 fusion protein (GLP-1(A8G)-LK-L13H13) withhuman GLP-1R (hGLP-1R) stably expressed in the Chinese hamster ovarycell line (solid line peak, marked with *) in comparison with theChinese hamster ovary cell line itself (dotted line peak).

FIG. 2 shows the reporter gene assay dose-responsive curves of GLP-1wild type (circles) and GLP-1(A8G)-LK-L13H13 (triangles) activatinghGLP-1R stably expressed in Chinese hamster ovary cell line.

FIG. 3 is the result of a mouse (ICR) glucose tolerance test, showingthe glucose tolerance of fasting mice 16 hr single i.p. injection ofGLP-1(A8G)-LK-L13H13 at 5 micrograms per mouse (square) and 15micrograms per mouse (triangles).

FIG. 4 is the result of a mouse (C57BL) glucose tolerance test, showingthe glucose tolerance of fasting mice 40 hr single i.p. injection ofGLP-1(A8G)-LK-L13H13 at 15 micrograms per mouse (triangles).

FIG. 5 is the blood glucose concentration-time curve of the type IIdiabetic mice (db/db mice), reflecting the blood glucose concentrationchange over the experiment period of the type II diabetic mice after asingle i.p. injection of GLP-1(A8G)-LK-L13H13 at a concentration of 10nmol/kg (inverted triangles).

FIG. 6 is the daily food intake-time curve of the type II diabetic mice(db/db mice), reflecting the mice daily food intake change of the typeII diabetic mice after i.p. injection of GLP-1(A8G)-LK-L13H13 at 10nmol/kg (inverted triangles), during the time period from 3 days beforeinjection of the fusion protein to 5 days after injection. FIG. 6 andFIG. 5 are the results of two parallel experiments.

SPECIFIC EMBODIMENTS OF THE INVENTION

Through the following specific embodiments in combination with thefigures, the technical solutions of the present invention are furtherillustrated.

In this invention, unless referred specifically, the employed rawmaterials, equipments and the like can all be purchased from the marketor are commonly used in the art. The methods of the followingembodiments, if not indicated specifically, are all conventional methodsin the art.

Step 1: Construction of Stable Antigen Cell Line for Immunization

CHO-DHFR minus cells are transferred into a 6-well plate and transfectedwith the pYS plasmid carrying hGLP-1R gene (see SEQ ID NO: 113 for thenucleotide sequence, and see SEQ ID NO: 114 for the amino acid sequence)after 24 hr culture. The medium is changed before transfection, and itis carried out by following the recommended transfection conditionsprovided by the manufacturer of Lipofectamine 2000 (Invitrogen). 48 hrafter transfection, the medium of the culture is replaced by thecomplete medium containing 10 nM MTX. The medium is changed every 3 daysfor about two weeks, until stable clones appear. The dispersed cellcolonies are detached from the plate and collected. After cells grow toabout 50% confluence, gradually increasing concentrations of MTX (up toa concentration of 10 μM MTX) are added for pressure selection. Theconstructed stable cell lines are tested by FACS analysis usingantibodies (Abcam) against hGLP-1R to identify cell clones afterpressure selection. There is a lot of hGLP-1R expression in the selectedCHO-DHFR-hGLP-1R cell membranes after MTX selection. Finally sixhigh-expression and stable cell lines of hGLP-1R are identified throughsubcloning.

Step 2: Preparation of Antibodies

Freund's adjuvant emulsified CHO-DHFR-hGLP-1R whole-cells are used at2×10⁶ cells/mouse dosage for subcutaneous injection into BALB/c mice(6-8 weeks). After 2 weeks, the immunity of the mice is boosted withincomplete Freund's adjuvant emulsified immunogen, and then once a week.The blood samples are collected from the clipped tail end andcentrifuged to collect the serum for detecting the serum titers by FACSanalysis. After the acceptable antibody titers are achieved, the miceare sacrificed and their spleen cells are harvested under asepticcondition. SP2/0 cells are collected at the logarithmic phase of growthwith 3 min centrifugation at 2000 rpm. The precipitation is resuspendedwith serum-free culture medium, then centrifuged and resuspended for asecond time, and counted. Spleen cells and SP2/0 cells are mixed atratio of SP2/0 cells:spleen cells ≥1:1, followed by 3 rounds ofwashing-centrifugation. After the precipitation from the lastcentrifugation is detached, 1 ml of the PEG-1350 (pre-warmed to 37° C.)is added drop wise (finished in 30 s), after pipette-mixing for 1 min,30 ml (pre-warmed to 37° C.) serum-free medium (Invitrogen) is addedslowly to terminate the PEG fusion. After 5 min centrifugation at 1500rpm, the cell pellets are resuspended and RPMI1640 (Invitrogen)containing HAT (sarcine, amethopterin and thymidine; Invitrogen) and 20%FBS (Bioind) is added as the fusion culture medium. 20000 spleen cellsand 5000 feeder layer cells in 100 μl volume are plated into each wellof 96-well plates. Fused hybridoma cells and feeder layer cells areco-cultured in 96-well plates with HAT selection to get rid of thenon-fused cells. After 10 days, the supernatant of the hybridoma cellsin the culture plates is collected for ELISA test.

Step 3: ELISA Screening of the Whole Cells

CHO-DHFR-hGLP-1R cells over-expressing hGLP-1R and CHO-DHFR minus cellsnot expressing hGLP-1R, were separately transferred into a 96-wellplate, and kept growing to 90% confluent. The supernatant of the culturemedium is removed and attached cells are washed twice with PBS, then 100μl 100% methanol is added to fix the cells for 10 min at 4° C. then 100μl freshly made 0.6% H₂O₂-PBS is added, and after incubation at roomtemperature for 20 min, the cells are washed twice with PBS. Afterblocking with PBS-1% BSA solution, the hybridoma supernatant is addedand incubated for 90 min at 4° C. After several washes, 100 μl of thesecondary antibody GxM-HRP-Fc (Sigma-Aldrich) (5000-times diluted) isadded into each well and incubated at 37° C. for 0.5 hr. After washingfor five times, 100 μl of TMB chromogenic substrate is added into eachwell and incubated at 37° C. for 15 min, and then 2M H₂SO₄ is added toterminate the reaction for reading of OD450 values. Positive control isthe mouse serum after immunization; negative control is the cell culturesupernatant. Hybridoma clones secreting anti-hGLP-1R antibody arescreened and the stable secretory cell lines against hGLP-1R areobtained after cloning. Lastly, antibody supernatant secreted byhybridoma is verified by FACS analysis.

Step 4: Flow Analysis (FACS) of the Supernatant of the PositiveHybridoma Cells

PBS containing 10 mM EDTA is used to detach and collect 10⁵ CHODHFR-hGLP-1R cells into a 1.5 ml EP tube. The supernatant is removedafter centrifugation and the negative control sample is resuspended witha loading buffer (PBS, 2% FBS). For positive control, 200 μl antibodysupernatant is added to resuspend the cells with incubation at roomtemperature; the cells are then centrifuged at 1500 rpm to remove thesupernatant, washed with loading buffer and centrifuged again. The cellsare resuspended with addition of FITC labeled goat anti-mousefluorescent antibody at 1:50 dilution (BD Pharmingen, 200 μl/well) andincubated at room temperature for 30 min in the dark. Supernatant isremoved after centrifugation, cells are washed with loading buffer,centrifuged again and resuspended with loading buffer for analysis. Thehybridoma supernatant and CHO-DHFR-hGLP-1R cells have specific binding:gray peak and dotted line peak are negative controls; the solid linepeak (marked with *), corresponding to the hybridoma supernatant, movesto the right obviously (FIG. 1).

Step 5: Cloning and Subcloning of Antibody Genes

Hybridoma cells secreting antibody are collected. Hybridoma mRNA isextracted according to the manufacturer protocol of QIAGEN mRNAextraction kit. Then the extracted mRNA is transcribed reversely intocDNA. The reverse transcription primers are specific primers for thelight and heavy chain constant regions of mouse, with the heavy chainreverse transcription primer being (5′-TTTGGRGGGAAGATGAAGAC-3′) (SEQ IDNO: 115), the light chain reverse transcription primers being(5′-TTAACACTCTCCCCTGTTGAA-3′) (SEQ ID NO: 116) and(5′-TTAACACTCATTCCTGTTGAA-3′) (SEQ ID NO: 117). RT-PCR reactionconditions are as following: 25° C. for 5 min, 50° C. for 60 min, and70° C. for 15 min. Reversely transcribed cDNA is diluted with 0.1 mM TEto 500 μl, added into the ultrafiltration centrifuge tube (AmiconUltra-0.5) and centrifuged at 2000 g for 10 min. The filtrate isremoved, 500 μl of 0.1 mM TE is added and centrifuged at 2000 g for 10min. The filtrate is removed and the preparation tube is placed ininversion to the new centrifugal tube, and centrifuged at 2000 g for 10min to obtain the purified cDNA. 10 μl of purified cDNA serves as thetemplate. Add 4 μl 5× tailing buffer, 4 μl dATP (1 mM) and 10 U terminaltransferase (Promega), mix uniformly and incubate at 37° C. for 5 minand at 65° C. for 5 min. The PolyA tail cDNA is used as templates andPCR is performed to amplify light and heavy chain variable region genesof antibodies. Upstream primers are all OligodT, with heavy chaindownstream primers being (5′-TGGACAGGGATCCAGAGTTCC-3′) (SEQ ID NO: 118)and (5′-TGGACAGGGCTCCATAGTTCC-3′) (SEQ ID NO: 119), and light chaindownstream primer being (5′-ACTCGTCCTTGGTCAACGTG-3′) (SEQ ID NO: 120).The PCR reaction conditions are as following: 95° C. for 5 min; 95° C.30 s, 56° C. for 30 s, 72° C. for 1 min, 40 cycles; and 72° C. for 7min. The PCR products are connected to the PMD 18-T vector forsequencing. The resulting sequences of the light and heavy chainvariable regions of the antibody after sequencing are listed in theattached Sequence Listing.

PCR primers are designed based on the sequenced DNA sequences of theantibody, thus the complete light chain, heavy chain signal peptides andvariable domains and mouse IgG1 constant region are connected withexpression vector pTM5.

Step 6: Transient Expression of Anti-GLP-1R Antibodies in HEK293Suspension Host Cell Line

The suspension HEK293 or CHO expressing cell line are inoculated to ashaker flask, and after 24 hr rotation at 37° C., the cells are readyfor transfection. Polyethylenimine (PEI) is used as a transfectionreagent during transfection, and its mixture with DNA is added into thecell culture. The mixing optimization ratio of PEI to DNA is 1:1 to 5:1.PEI/DNA mixture treated cells is rotated for more than 96 hr at 37° C.to express the antigen binding protein, meanwhile 0.5% of tryptone isadded into the cell culture as the source of amino acids required byexpression, and finally the cell supernatant is collected for thepurification and separation of the antigen binding protein.

Step 7: Antibody Humanization and Optimization

First of all, the sequences of light and heavy chain variable regions ofthe screened mouse antibody are aligned with the homologous antibodies,using NCBI online antibody variable region sequence alignment tool (IgBlast) to search the germline gene sequences of a humanized antibody (IgGermline Gene sequence) homologous to the selected antibodies variableregion sequence for humanization, and the humanized gene sequence withhighest homology except CDR sequences is used as template for CDRgrafting to get the humanized antibody variable region sequences and tosynthesize humanized antibody light and heavy chain genes. According tothe sequence, PCR primers are designed and at the 5′ end and 3′ end ofthe synthetic sequence, appropriate restriction enzyme sites areintroduced. By PCR, the humanized antibody variable regions areamplified and then combined with the human IgG2 or IgG4 constant regionsequence to obtain the whole recombinant humanized antibody sequence.The expression of the recombinant antibodies is achieved according tostep 6, and its affinity towards GLP-1R is verified by FACS analysis asdescribed in step 4. The best humanized antibody candidate retainingaffinity towards GLP-1R is selected from the group thereof, and by meansof site-specific mutagenesis, its variable region sequence is furtherimproved for better affinity towards GLP-1R.

Step 8: Cloning and Subcloning of Genes of the Humanized Fusion Proteinof GLP-1

Optimized humanized antibody is fused with GLP-1 or its derivativesequence, at the N-terminal and C-terminal of the light chains, to formthe GLP-1 fusion protein, and the sequences of the two are connected bya peptide linker sequence (LK). Nucleotide sequence of the signalpeptide-GLP-1 peptide linker is synthesized by Genscript BiotechnologyCO., LTD. Using the synthetic gene as the template, PCR amplifies thesequence of the part “signal peptide-GLP1-linker”, with PCR upstreamprimer being (5′-CCACCATGGACTTTGGGCTGAGC-3′) (SEQ ID NO: 121), PCRdownstream primer being (5′-AGAGCCGGTGGCAGAGCCAG-3′) (SEQ ID NO: 122).The PCR reaction conditions are as following: 95° C. for 5 min; 95° C.for 30 s, 56° C. for 30 s, 72° C. for 30 s, 35 cycles; 72° C. for 7 min.In addition, using the nucleotide sequence of the humanized antibody astemplate, the sequence of the antibody part of the fusion proteinsequence is amplified.

The PCR upstream primer is(5′-CTGGCTCTGCCACCGGCTCTGCCATCCAGATGACCCAGTCTCC-3′) (SEQ ID NO: 123) andthe PCR downstream primer is (5′-ACACTCTCCCCTGTTGAAGCTC-3′) (SEQ ID NO:124). The PCR reaction conditions are as following: 95° C. for 5 min;95° C. for 30 s, 56° C. for 30 s, 72° C. for 1 min, 35 cycles; 72° C.for 7 min. Then through overlapping PCR, the part “signalpeptide-GLP-1-peptide linker” of the nucleic acid sequence of the fusionprotein is connected with the antibody part, introducing two restrictionenzyme sites Nhe1 and Not1 to both ends of the primers, and thuscomplete fusion protein sequence and the expression vector pTM5 arelinked together. Overlapping PCR upstream primer is(5′-CCGGCTAGCCACCATGGACTTTGGGCTGAGC-3′) (SEQ ID NO: 125) and thedownstream primer is (5′-AGTGCGGCCGCTCAACACTCTCCCCTGTTGAAGCTC-3′) (SEQID NO: 126). The PCR reaction conditions are as following: 95° C. for 5min; 95° C. for 30 s, 56° C. for 30 s, 72° C. for 1 min, 35 cycles; 72°C. for 7 min. The PCR products are connected to the PTM5 vector and thenare confirmed by sequencing.

Step 9: Transient Expression of GLP-1 Fusion Protein in HEK293Suspension Host Cell Line

HEK293 suspension cells are inoculated into a shaker flask and theculture medium is changed before transfection. The vectors containingfusion protein light/heavy chain gene at the concentration of 0.5 to 1.5μg/ml based on the total amount of DNA are mixed with 1.5 to 7.5 μg/mlof polyethylenimine (PEI), and after standing for 15-25 minutes themixture is added into the cell culture medium. After 24 hr, 0.5-1% ofTrypton N1 is added into the cell culture medium. The supernatantscontaining the secreted GLP-1 fusion protein are harvested afterculturing for 5-10 days.

Step 10: Stable Expression of GLP-1 Fusion Protein in CHO SuspensionHost Cell Line.

CHO suspension cells are inoculated into a 6-well plate, and thetransfection of the expression vector of the fusion protein isimplemented by the transfection conditions in step 1. After 48 hr, 300mg/ml of hygromycin (heavy chain) and 6 mg/ml puromycin (light chain)are added for co-selection. After apoptosis occurred in quantity(mortality rate >90%), antibiotic concentration is gradually reduced toallow the remaining cells to recover and transferred into a shaker flaskfor culture expansion, then the expression of the antibody in thesupernatant is confirmed. Afterwards, half of antibiotic concentrationsin medium is maintained to allow the stable expression of GLP-1 fusionprotein of the cells.

Step 11: Purification and Preparation of GLP-1 Fusion Protein from theCell Culture Supernatant

Cells are removed from the culture after centrifugation, and thesupernatant runs through G protein coupled affinity chromatographycolumn. The expressed GLP-1 fusion protein is eluted from thechromatography column using eluent with pH around 2.5-3.5. The low pHvalue of the eluent is neutralized immediately with the neutralizedbuffer provided in the elution tube. The protein solution is collectedafter elution and is then dialyzed against PBS.

Step 12: Reporter Gene Assay Test of GLP-1 Fusion Protein for itsFunction In Vitro Activation of GLP-1R (See FIG. 2).

The CHO-DHFR minus cells co-expressing hGLP1R-CRE-Luciferase areinoculated into a 96-well cell culture plate with 20000 cells per well,and cultured at 37° C. overnight. The next day the culture supernatantis removed. The cell surfaces are washed twice with serum free mediumand residual liquid is removed as well, then adding 100 μl serum freemedium for dilution and purification of antibodies or GLP-1 andincubated at 37° C. for 4 hr. After the stimulation, 100 μl Bright Glochemiluminescence substrate of Promega is added. Finally the celllysates are transferred into a white 96-well plate, and the relativeluminous intensity is recorded in SpectraMax L microplate reader ofMolecular Devices.

Step 13: Glucose Tolerance Test of Fasting ICR Mice.

Glucose tolerance of mice in vein is determined to evaluate the efficacyof the GLP-1 fusion proteins (preferably, antibody GLP-1(A8G)-LK-L13H13) disclosed in the present patent. There are four groupsof mice, and each group contains at least three to five mice. Group I isthe control group, received an intraperitoneal injection of same volumeof PBS. Group II received a single intraperitoneal injection of 15 μgper mouse. Group III received a single intraperitoneal injection of 5 μgper mouse. After injection, mice are fasted for 6-16 hr, and after thefasting is complete, the blood samples of mice are taken to determinethe basal blood glucose concentration. Then the mice are forced with agavage of glucose at the concentration of 1.5 g/kg, and 15, 30, 60 and90 min after the gavage, the blood samples are taken to determine bloodglucose concentration, as shown in FIG. 3.

Step 14: Glucose Tolerance Test of GLP-1 Fusion Protein in Mice (C57BL)for Long-Term (40 hr) Efficacy.

Glucose tolerance of mice in vein is determined to evaluate thelong-term efficacy of the GLP-1 fusion proteins (preferably, antibodyGLP-1 (A8G)-LK-L13H13) disclosed in the present patent. There are twogroups of mice, and each group contains at least three to five mice.Group I is the control group, received an intraperitoneal injection ofsame volume of PBS. Group II received a single intraperitoneal injectionof 15 μg per mouse. 24 hr after injection, mice are fasted for 16 hr,and after the fasting is complete, the blood samples of mice are takento determine the basal blood glucose concentration. Then the mice areforced with a gavage of glucose at the concentration of 1.5 g/kg, and15, 30, 60 and 90 min after the gavage, the blood samples are taken todetermine blood glucose concentration, as shown in FIG. 4.

Step 15: Study on the Long-Term (72 hr) Effects of a GLP-1 FusionProtein on Lowering the Blood Glucose Concentration of Type II DiabeticMice (Db/Db Mice).

The blood glucose concentration of type II diabetic mice are determinedat different time points to evaluate the long-term efficacy of the GLP-1fusion proteins (preferably, antibody GLP-1 (A8G)-LK-L13H13) disclosedin the present patent in lowering the blood glucose concentration oftype II diabetic mice. There are two groups of mice, and each groupcontains at least six mice. Before the start of the injection, the bloodsamples of mice are taken to determine the basal blood glucoseconcentration. Later, Group I (the control group), received anintraperitoneal injection of same volume of PBS. Group II received asingle intraperitoneal injection of the GLP-1 fusion protein at aconcentration of 10 nmol/kg. 1, 4, 6, 24, 48 and 72 hr after injection,the blood samples thereof are taken respectively to determine the bloodglucose concentration of the two groups of mice, as shown in FIG. 5.

Step 16: Study on the Long-Term (120 hr) Effects of a GLP-1 FusionProtein on the Reduction of the Daily Food Intake in Type II DiabeticMice (Db/Db Mice).

The food intake changes of type II diabetic mice are determined toevaluate the long-term efficacy of the GLP-1 fusion proteins(preferably, antibody GLP-1 (A8G)-LK-L13H13) disclosed in the presentpatent in reducing the food intake level of type II diabetic mice. Thisstep is carried out along with step 15 on the same batch of mice insynchronization. There are two groups of mice, and each group containsat least six mice. From 3 days before the injection of step 15 to 5 daysafter injection (120 hr), every morning and at the same time, the dailyfood intake of each group of mice was weighed, as shown in FIG. 6.

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the claimed embodiments, and are not intended to limit thescope of what is disclosed herein. Modifications that are obvious topersons of skill in the art are intended to be within the scope of thefollowing claims.

What is claimed is:
 1. A method of treating non-insulin-dependentdiabetes in a subject, comprising administering to the subject atherapeutically effective amount of a GLP-1 fusion protein comprising aGLP-1 and an antibody specifically binding to GLP-1R, wherein theantibody comprises: heavy chain CDR1 amino acid sequence: SEQ ID NO: 12,heavy chain CDR2 amino acid sequence: SEQ ID NO: 19, heavy chain CDR3amino acid sequence: SEQ ID NO: 27, light chain CDR1 amino acidsequence: SEQ ID NO: 37, light chain CDR2 amino acid sequence: SEQ IDNO: 45, and light chain CDR3 amino acid sequence: SEQ ID NO:
 53. 2. Themethod of claim 1, wherein the antibody comprises light chain variableregion sequence: SEQ ID NO: 101 or SEQ ID NO: 105; and heavy chainvariable region sequence: SEQ ID NO: 75 or SEQ ID NO:
 79. 3. The methodof claim 1, wherein the antibody comprises a light chain variable regionsequence encoded by polynucleotide sequence: SEQ ID NO: 100 or SEQ IDNO: 104; and a heavy chain variable region sequence encoded bypolynucleotide sequence: SEQ ID NO: 74 or SEQ ID NO:
 78. 4. The methodof claim 1, wherein the antibody comprises light chain variable regionsequence: SEQ ID NO: 101; and heavy chain variable region sequence: SEQID NO:
 75. 5. The method of claim 1, wherein the antibody comprises alight chain variable region sequence encoded by polynucleotide sequence:SEQ ID NO: 100; and a heavy chain variable region sequence encoded bypolynucleotide sequence: SEQ ID NO:
 74. 6. The method of claim 1,wherein the antibody comprises light chain variable region sequence: SEQID NO: 105; and heavy chain variable region sequence: SEQ ID NO:
 79. 7.The method of claim 1, wherein the antibody comprises a light chainvariable region sequence encoded by polynucleotide sequence: SEQ ID NO:104; and a heavy chain variable region sequence encoded bypolynucleotide sequence: SEQ ID NO:
 78. 8. The method of claim 1,wherein the antibody further comprises an amino acid sequence selectedfrom: (a) light chain constant region amino acid sequence: SEQ ID NO106; (b) light chain constant region amino acid sequence: SEQ ID NO 107;(c) heavy chain constant region amino acid sequence: SEQ ID NO 108; (d)heavy chain constant region amino acid sequence: SEQ ID NO 109; (e)light chain constant region amino acid sequence: SEQ ID NO 106 and heavychain constant region amino acid sequence: SEQ ID NO 108; (f) lightchain constant region amino acid sequence: SEQ ID NO 107 and heavy chainconstant region amino acid sequence: SEQ ID NO 108; (g) light chainconstant region amino acid sequence: SEQ ID NO 106 and heavy chainconstant region amino acid sequence: SEQ ID NO 109; and (h) light chainconstant region amino acid sequence: SEQ ID NO 107 and heavy chainconstant region amino acid sequence: SEQ ID NO
 109. 9. The method ofclaim 1, wherein the antibody is a humanized antibody.
 10. The method ofclaim 1, wherein the GLP-1 comprises an amino acid sequence selectedfrom SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:
 127. 11. The method of claim 1, wherein the GLP-1comprises amino acid sequence SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:5.
 12. The method of claim 1, wherein the GLP-1 comprises amino acidsequence SEQ ID NO:
 5. 13. The method of claim 1, wherein the GLP-1 isfused with a light chain of the antibody via a peptide linker.
 14. Themethod of claim 13, wherein the peptide linker comprises amino acidsequence SEQ ID NO: 110, SEQ ID NO: 111, or SEQ ID NO:
 112. 15. Themethod of claim 1, wherein the GLP-1 is fused with the light chainand/or heavy chain of the antibody in the form of N′—R1-L-R2-C′,N′—R2-L-R1-C′ or N′—R2-R1r-C′, wherein: L is a peptide linker,comprising a full-length, partial, or repeated amino acid sequenceselected from SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112; R1 isan amino acid sequence of GLP-1; R1r is a reverse amino acid sequence ofGLP-1; R2 is an amino acid sequence of the light chain or heavy chain ofthe antibody; C′ represents a carboxyl residual terminal of the GLP-1fusion protein polypeptide; and N′ represents an amino residual terminalof the GLP-1 fusion protein polypeptide.
 16. A method of treatingobesity in a subject, comprising administering to the subject atherapeutically effective amount of a GLP-1 fusion protein comprising aGLP-1 and an antibody specifically binding to GLP-1R, wherein theantibody comprises: heavy chain CDR1 amino acid sequence: SEQ ID NO: 12,heavy chain CDR2 amino acid sequence: SEQ ID NO: 19, heavy chain CDR3amino acid sequence: SEQ ID NO: 27, light chain CDR1 amino acidsequence: SEQ ID NO: 37, light chain CDR2 amino acid sequence: SEQ IDNO: 45, and light chain CDR3 amino acid sequence: SEQ ID NO:
 53. 17. Amethod of treating overweight in a subject, comprising administering tothe subject a therapeutically effective amount of a GLP-1 fusion proteincomprising a GLP-1 and an antibody specifically binding to GLP-1R,wherein the antibody comprises: heavy chain CDR1 amino acid sequence:SEQ ID NO: 12, heavy chain CDR2 amino acid sequence: SEQ ID NO: 19,heavy chain CDR3 amino acid sequence: SEQ ID NO: 27, light chain CDR1amino acid sequence: SEQ ID NO: 37, light chain CDR2 amino acidsequence: SEQ ID NO: 45, and light chain CDR3 amino acid sequence: SEQID NO:
 53. 18. The method of claim 16, wherein the antibody compriseslight chain variable region sequence: SEQ ID NO: 101 or SEQ ID NO: 105;and heavy chain variable region sequence: SEQ ID NO: 75 or SEQ ID NO:79.
 19. The method of claim 16, wherein the antibody comprises a lightchain variable region sequence encoded by polynucleotide sequence: SEQID NO: 100 or SEQ ID NO: 104; and a heavy chain variable region sequenceencoded by polynucleotide sequence: SEQ ID NO: 74 or SEQ ID NO:
 78. 20.The method of claim 16, wherein the antibody comprises light chainvariable region sequence: SEQ ID NO: 101; and heavy chain variableregion sequence: SEQ ID NO:
 75. 21. The method of claim 16, wherein theantibody comprises a light chain variable region sequence encoded bypolynucleotide sequence: SEQ ID NO: 100; and a heavy chain variableregion sequence encoded by polynucleotide sequence: SEQ ID NO:
 74. 22.The method of claim 16, wherein the antibody comprises light chainvariable region sequence: SEQ ID NO: 105; and heavy chain variableregion sequence: SEQ ID NO:
 79. 23. The method of claim 16, wherein theantibody comprises a light chain variable region sequence encoded bypolynucleotide sequence: SEQ ID NO: 104; and a heavy chain variableregion sequence encoded by polynucleotide sequence: SEQ ID NO:
 78. 24.The method of claim 16, wherein the antibody further comprises an aminoacid sequence selected from: (a) light chain constant region amino acidsequence: SEQ ID NO 106; (b) light chain constant region amino acidsequence: SEQ ID NO 107; (c) heavy chain constant region amino acidsequence: SEQ ID NO 108; (d) heavy chain constant region amino acidsequence: SEQ ID NO 109; (e) light chain constant region amino acidsequence: SEQ ID NO 106 and heavy chain constant region amino acidsequence: SEQ ID NO 108; (f) light chain constant region amino acidsequence: SEQ ID NO 107 and heavy chain constant region amino acidsequence: SEQ ID NO 108; (g) light chain constant region amino acidsequence: SEQ ID NO 106 and heavy chain constant region amino acidsequence: SEQ ID NO 109; and (h) light chain constant region amino acidsequence: SEQ ID NO 107 and heavy chain constant region amino acidsequence: SEQ ID NO
 109. 25. The method of claim 16, wherein theantibody is a humanized antibody.
 26. The method of claim 16, whereinthe GLP-1 comprises an amino acid sequence selected from SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:127.
 27. The method of claim 16, wherein the GLP-1 comprises amino acidsequence SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:
 5. 28. The method ofclaim 16, wherein the GLP-1 comprises amino acid sequence SEQ ID NO: 5.29. The method of claim 16, wherein the GLP-1 is fused with a lightchain of the antibody via a peptide linker.
 30. The method of claim 29,wherein the peptide linker comprises amino acid sequence SEQ ID NO: 110,SEQ ID NO: 111, or SEQ ID NO:
 112. 31. The method of claim 16, whereinthe GLP-1 is fused with the light chain and/or heavy chain of theantibody in the form of N′-R1-L-R2-C′, N′-R2-L-R1-C′ or N′-R2-R1r-C′,wherein: L is a peptide linker, comprising a full-length, partial, orrepeated amino acid sequence selected from SEQ ID NO: 110, SEQ ID NO:111, and SEQ ID NO: 112; R1 is an amino acid sequence of GLP-1; R1r is areverse amino acid sequence of GLP-1; R2 is an amino acid sequence ofthe light chain or heavy chain of the antibody; C′ represents a carboxylresidual terminal of the GLP-1 fusion protein polypeptide; and N′represents an amino residual terminal of the GLP-1 fusion proteinpolypeptide.